Proximal colon distension induces Fos expression in oxytocin-, vasopressin-, CRF- and catecholamines-containing neurons in rat brain

Abstract

Little is known about the chemical coding of the brain neuronal circuitry activated by nociceptive signals of visceral origin. We characterized brain nuclei activated during isovolumetric phasic distension of the proximal colon (10 ml, 30 s on/off for 10 min) in conscious male rats, using Fos as a marker of neuronal activation and dual immunohistochemistry to visualize co-localization of Fos expression and oxytocin (OT), arginine-vasopressin (AVP), corticotrophin-releasing factor (CRF) or tyrosine hydroxylase (TH). Proximal colon distension, compared with sham distension, induced a robust increase in Fos-like immunoreactive (IR) neurons in the paraventricular nucleus (PVN), supraoptic nucleus (SON) and accessory neurosecretory nuclei of the hypothalamus, nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), and to a lower extent, in the locus coeruleus (LC) and Barrington nucleus. Fos-IR neurons in the PVN after colon distension were identified in 81% of OT-IR, 18% AVP-IR and 16% CRF-IR neurons, while in the SON it represented 36% of OT-IR and 16% AVP-IR. Catecholaminergic cell groups in the pons (LC) and medulla (VLM, NTS) were also activated by proximal colon distension. Of the TH-IR neurons in VLM and NTS, 74% and 42% respectively were double labeled. These results indicate that colon distension stimulates OT-, AVP- and CRF-containing hypothalamic neurons, likely involved in the integration of colonic sensory information to modulate autonomic outflow and pain-related responses. Activation of medullary catecholaminergic centers might reflect the afferent and efferent limbs of the functional responses associated to visceral pain.

Fig. 1

Double immunohistochemical staining of Fos- and TH-IR in the NTS (A and B) and VLM (C and D) and cell count (cells per section) in the same areas (E) 60 min after phasic proximal colon distension for 10 min (B and D) or sham distension (A and C) in conscious rats. Fos-IR is localized in nuclei and appears as dark blue-black dots and TH-IR is found in the cytoplasm as brown color. Arrow: TH-IR; arrowhead: Fos-IR; arrowheads: Fos/TH-IR; AP: area postrema; DMV: dorsal motor nucleus of the vagus. Data are mean±SEM; n=5; *P<0.05 vs sham distension group.

Fig. 2

Double immunohistochemical staining of Fos- and TH-IR in the pons 60 min after phasic proximal colon distension for 10 min (B and D) or sham distension (A and C) in conscious rats. A and B show the locus coeruleus (LC) while C and D show the Barrington nucleus (Bar). Me5: mesencephalic nucleus of trigeminal nerve (5^th cranial nerve; the staining in panel D is non-specific).

Fig. 3

Double immunohistochemical staining of Fos- and OT-IR (A-D) or AVP-IR (E and F) in the PVN of the hypothalamus 60 min after phasic proximal colon distension for 10 min (A-C and E) or sham distension (D and F) in conscious rats. The inserts in A and E are magnification of the framed areas. A: Fos- and OT-IR double labeling in the medial magnocellular (mm) division and dorsal cap (dc). B and D: Fos- and OT-IR double-labeled neurons in the lateral magnocellular (lm), ventral (v), medial parvocellular (mp), dorsal cap (dc) and periventricular (pe) divisions of the PVN. C: Fos- and OT-IR double labeling in the posterior (p) PVN. Note the almost complete absence of double-stained cells. E and F: Fos- and AVP-IR double labeling in the PVN. Bar in B indicates the same scale in all panels while the scale in A corresponds to the two inserts.

Fig. 4

Cell counts (cells per section) of Fos- and OT-IR or AVP-IR in the PVN 60 min after phasic proximal colon distension for 10 min in conscious rats. Data are mean±SEM; n=5; *P<0.05 vs sham distension group.

Fig. 5

Double immunohistochemical staining of Fos- and OT-IR (A and B) or AVP-IR (C and D) in the SON of the hypothalamus 60 min after phasic proximal colon distension for 10 min (B and D) or sham distension (A and C) in conscious rats. Bar = 100 μm. E: Cell counting (cells per section) in the SON.

Fig. 6

Double immunohistochemical staining of Fos- and OT-IR (A-D) or AVP-IR (E) in the accessory neurosecretory nuclei of the hypothalamus 60 min after phasic proximal colon distension for 10 min (A-C and E) or sham distension (D) in conscious rats. A: parafornical nucleus; B-E: lateral hypothalamus. Double-labeled neurons were often found surrounding the blood vessels (A, B and E).

Fig. 7

Double immunohistochemical staining of Fos- and CRF-IR in the PVN. Animals were treated with icv colchicine 24 h before the distension protocol. A: sham distension; B: proximal colon distension.